DESCRIPTION: Enterococci, predominantly Enterococcus faecalis, cause 5-15 percent of cases of infective endocarditis and are the second most common pathogens isolated from hospital acquired infections. Antimicrobial resistances may be partly responsible for this latter role and certainly make it more difficult to successfully treat patients who are infected. The therapeutic dilemmas posed by enterococci have drastically worsened over the past decade with the emergence of high-level resistance to all aminoglycosides, beta-lactamase and non-beta-lactamase mediated resistance to penicillins, and resistance to vancomycin, the most devastating of these traits. Because of our rapidly declining antimicrobial options, it seems appropriate to increase investigative efforts to delineate pathogenic mechanisms of enterococci. Perhaps by understanding the mechanisms, other therapeutic or preventative modalities can eventually be developed, and, indeed, it is now critical that such modalities be pursued. In our current proposal, we have developed the genetic methods to create targeted knock-out mutations in E. faecalis genes and have obtained sequence from inactivated genes and from subclones of our large cosmid clones, including three encoding immunoreactive antigens, and have characterized a number of clinical isolates and patient sera. In the competitive renewal application, we plan to further investigate E. faecalis genes encoding in vivo expressed antigens. This will be done with 43 different (as evidenced by different locations on the chromosome or restriction endonuclease digestion analysis) immunopositive clones. Based on preliminary results to date, it is estimated that from these 43 immunopositive clones, we may identify 2 or 3 antigen encoding genes per clone. Depending on the number of bands seen on Western blots of clones, characterization of clones will be done either by transposon mutagenesis to inactivate immunoreactivity or by subcloning Dnase I generated smaller fragments (from our 30-40 kb inserts) that still encode immunopositivity. DNA sequence will be generated using primers based on the transposon ends or the vectors used for subcloning, followed by database searches for possible homologies. Targeted "cross back" insertion mutants in E. faecalis of "high priority" genes will then be made, e.g., those with homologies to genes implicated in pathogenesis of other organisms. E. faecalis isogeneic except for specific genes will then be compared in animal model(s) and biologic assays to evaluate the consequences of gene inactivation. Our hypotheses are that among in vivo expressed antigens (at least some of which are likely to have a surface domain) are ones which are involved in production of disease, and that preventative or therapeutic modalities can be rationally designed based on knowledge about these proteins. Results from this work may provide a foundation for development of non-antibiotic methods (e.g., passive or active immunization) to prevent, control, or combat E. faecalis infections, and may identify new targets for traditional antibiotic development strategies.